Helicobacter pylori is a bacterium that infects the stomach lining (or gastric mucosa) of perhaps half the world's population. Spiral organisms were first microscopically observed in human gastric mucosa in 1906. However, H. pylori was not successfully cultured until 1982. Infection with the organism is usually chronic, and results in continuing inflammation of the gastric mucosa. The infection is often asymptomatic. However, in association with other cofactors, a proportion of infected people go on to develop sequelae including peptic ulceration of the stomach or duodenum, gastric adenocarcinomas and gastric lymphomas. Peptic ulcer treatment studies have shown that cure of H. pylori infection is associated with a dramatic reduction in the relapse rate of this usually chronic disease. Long term infection with H. pylori leads to the development of chronic atrophic gastritis, which has long been recognised as a precursor lesion in the development of gastric cancer. Thus a number of studies have now linked preceding H. pylori infection with an increased risk of developing gastric cancer. Therefore eradication of current infection and prevention of new infection with this organism has the potential to significantly reduce the incidence of diseases that result in considerable morbidity and mortality.sup.1,2.
Infection with H. pylori is difficult to treat. Current experimental therapies for treating the infection have problems with efficacy and significant levels of adverse effects. There are no prophylactic measures available. A solution to both the prevention and treatment of H. pylori infection would be the development of an immunogenic preparation that, as an immunotherapeutic, treated established infections, and as a vaccine, prevented the establishment of new or recurrent infections. Such a preparation would need to induce effective immune responses to protective antigens, while avoiding inducing responses to self antigens or other potentially harmful immune responses. This may be achieved by identifying the specific protective component or components and formulating immunotherapeutic or vaccine preparations including these component(s).
The identification of such protective components of an organism, is often accomplished through the use of an animal model of the infection. Initially, H. pylori did not naturally infect laboratory animals. However, an animal model of human H. pylori infection has been developed using a closely related organism, H. felis, and specific pathogen free (SPF) mice.sup.3. These organisms are able to colonise the gastric mucosa of SPF mice, where they establish a chronic infection with many of the features of H. pylori infection in humans. H. felis infection in the mice induces a chronic gastritis and a raised immune response. As in the human case, this response is not effective in curing the infection.
This model has been used to demonstrate that oral treatment of H. felis infected mice with a preparation containing disrupted H. pylori cells and cholera toxin as a mucosal adjuvant, can cure a significant portion of infected mice.sup.4. This effect is likely to be mediated through an immune response to a cross-reactive antigen possessed by each of the closely related species.
In working by the inventors leading to the present invention, these cross-reactive antigens were recognised by performing a Western blot using H. pylori disrupted cells as the antigen, and probing the blot with serum from mice immunised with H. felis and cholera toxin adjuvant. Sections of membrane containing proteins recognised as cross-reactive were removed from the membrane, the proteins bound to them were eluted, and their N-terminal amino acid sequence determined by microsequencing.
The N-terminal amino acid sequence of one of the two proteins that successfully yielded sequence data closely matched the previously published sequence of the microbial enzyme, urease.sup.5. This enzyme has already been shown to be a protective antigen when used in a vaccine to prevent infection.
The N-terminal amino acid sequence of the other protein closely matched the previously published N-terminal sequence of the microbial enzyme, catalase.sup.6. This enzyme has not previously been shown to be a protective antigen of H. pylori.
International Patent Application No. PCT/FR95/00383 (Publication No. WO 95/27506) in the name Pasteur Merieux Serums et Vaccins, published Oct. 19, 1995, discloses an H. pylori immunising composition, based on proposed use of H. pylori catalase in substantially purified form as an immunising substance useful for prophylactic or therapeutic purposes. It is suggested that the catalase could be obtained either by extraction from H. pylori (using the purification method of Hazell et al..sup.10) or by recombinant means. The disclosure contains no supporting data showing efficacy of H. pylori catalase in use as an immunising substance, nor is there any supporting disclosure or teaching of the preparation of H. pylori catalase by recombinant means or of the efficacy of recombinant catalase in use as an immunising substance.
Recently, an H. pylori (Sydney strain)/mouse model of human H. pylori infection has been developed and used by the present inventors to confirm that catalase, in particular recombinant catalase, has utility as a protective antigen.